Septic shock remains as a major healthcare problem both in the U.S. and worldwide and, while the underlying mechanisms that contribute to disease are becoming increasingly well understood, there remain challenges in development of effective therapeutic interventions. The host response to potent inflammatory microbe- associated stimuli such as lipopolysaccharide (LPS), typically results in development of systemic inflammation, frequently referred to as a syndrome of mediator excess. This response is typically followed by a transient state of relative refractoriness to further stimulation, historically termed tolerance. Recent results from this laboratory have contributed to the development of the novel concept that the innate immune response macrophage ubiquitin-proteasome pathway functions as a key regulator of LPS-induced signaling to regulate the inflammatory response through TLR4 in murine cells. The precise mechanisms that determine whether an inflammatory stimulus induces a proinflammatory cytokine response or a state of unresponsiveness/tolerance have not been dissected in human monocytes. Of particular potential importance is the status of the host innate immune in patients with SIRS/sepsis relative to normal individuals. Consequently, there is a critical need to delineate the intricate relationship between proteasome activity and differential regulation of signaling pathways in human monocytes/macrophages in health and disease, as new knowledge about these processes can ultimately be expected to facilitate development of innovative treatment strategies that target the proteasome. The central hypothesis is that development of inflammation vs. tolerance in human monocytes/macrophages in response to LPS is dictated by the profile of proteasomal protease and that these processes are disrupted during the development of SIRS/sepsis. Three Specific Aims, all based on strong preliminary data are proposed: 1. Identify mechanisms of selective proteasomal subunit-dependent mediation of LPS-induced inflammation. 2. Determine mechanism(s) by which selective proteasome subunits contribute to LPS-induced tolerance/hyporesponsiveness. 3. Develop in vitro strategies to correct aberrant TLR responsiveness in human monocytes from SIRS/ sepsis patients. It is anticipated that the successful completion of the studies outlined in this proposal will ultimately contribute to development and implementation of therapeutic strategies to reduce morbidity and mortality of septic shock and, possibly, other cytokine-mediated diseases.